This is a competitive renewal of NIH grant DK33201-23 entitled Insulin Receptor Substrates and Insulin Action which focuses on studying the role of phosphorylation, in particular of the insulin receptor substrates and their partners, in the mechanism of insulin action. Over the past 5 years we have studied the similarities, differences and complementary roles of various insulin receptor substrates (IRS) in coupling the insulin/IGF-1 receptors to downstream effectors through the creation and characterization of cell lines and mice with knockout or knockdown of the IRS proteins. We have demonstrated how different IRS proteins play differential roles in insulin action on glucose transport, mitosis, apoptosis, differentiation of adipocytes, and gene expression. We have demonstrated in vitro and in vivo how insulin signaling pathways for glucose and lipid metabolism diverge and involve differential signaling through Akt and atypical PKC. We have identified and characterized at a molecular level a number of molecules and pathways that modify IRS signaling. We have shown how different insulin resistant states and background genes further modify these primary defects. This work has led us to develop a new model of the insulin signaling network in which there are critical nodes of signal divergence and regulation. In this network, the different IRS proteins constitute a critical node and mediate different signaling pathways, thus providing complementary information to different downstream actions of insulin. Genetic and acquired alterations in these pathways can lead to insulin resistant states, such as type 2 diabetes and metabolic syndrome, and background genes and environmental factors further modulate this response. Indeed, by a combination of genetics, genomics and proteomics, we have identified kinase C (PKC) 4 as the first of the candidate background modifier genes that could contribute to these major differences in insulin sensitivity between mouse strains. The specific aims for the next five years are to: 1) Define the native IRS signaling complexes and their interacting downstream partners in intact cells in culture and tissues in vivo using proteomic and phosphoproteomic approaches, and to determine how these pathways are altered in cells or mice with deletion of specific IRS proteins and insulin resistant states. 2) Define the role of specific IRS proteins and their downstream partners in control of gene expression in response to insulin and IGF-1, with the ultimate goal of integrating these changes in gene expression with those defined by proteomics and phosphoproteomics and the biological responses to have an integrated picture of the molecular mechanisms of insulin action and insulin resistance. 3) Determine the physiological role and mechanisms by which PKC4 might modify insulin action at a molecular level, and continue to use the combination of genetics, genomics, and proteomics to identify other background genetic modifiers of insulin resistance. PUBLIC HEALTH RELEVANCE: This grant focuses on studying the intracellular signaling pathways involved in insulin action and how these pathways are altered in disease such as diabetes and obesity. Using a combination of genetic, genomic and proteomic approaches, we have developed a new model of the insulin signaling network, identified many of its components, and begun some of the mechanisms underlying these common metabolic disorders.